EC Line and the CL-10 Plus System
Product Training
The EC- Line Product Range
• CL-10 PLUS
Instrument using the proprietary differential-pH
technology.
• EC- Line
Reagents. EC-Line is the range of reagents and
diagnostics to be used with CL-10 instrument.
The differential pH principle
Let’s study the differential pH principle using
the Urea Test in Milk.
The components of the CL-10 Plus System
Instrument:
• Two capillary glass electrodes to
measure pH
• Mixing chamber
• Peristaltic pumps
• Tubings that faciliate transfer of
reagents and samples in the
instrument
Kits and supplies (EC-Line):
(Urea Milk)
-
Polif solution (Wash Solution)
Regenerating Solution
Strong regenerating solution
(Urea Kit) Reagents for the
enzymatic reaction. Shelf life: 12
months
Basic components of the instrument
Left: Where solutions/samples
get loaded into the instrument
Right: Where solutions/samples exit and
enter the waste bottle
Waste
Bottle
Resting: Polif
Solution
Testing: R1
Diluent
Resting: Distilled Water
Testing: Urease Enzyme
Mixing chamber
Where milk samples are
pipetted into
Pipetting into the reaction chamber
Technique for pipetting into the
reaction chamber.
1) Load sample into pipette
2) Insert pipet into reaction
chamber
3) Wait until you can feel the stirbar
stirring
4) Depress pipette all the way to
release sample and keep it
depressed as you lift the tip out
of the chamber
*This prevents fluid from being
drawn out of the reaction
chamber, affecting the reaction
*Pipette tips are re-usable, wipe the
outside of the pipette gently
before inserting new samples
The Urea Reaction
• Urease is an enzyme that catalyzes the
hydrolysis of urea into carbon dioxide and
ammonia.
The Reaction Urease
Urea + 3 H2O  2NH4+ + CO2 + 2 OHH2O + CO2  HCO3- + H+
As the reaction proceeds, the level of H+
will increase, the pH will decrease.
As indicated in the reaction, the number of
H+ ion is directly proportional to
concentration of Urea.
Injecting Sample
Micropipette
Containing Sample
pH
D1
D2-D1
dpH=0
D2
Time (s)
D2
Vial containing
Enzyme (Urease)
Electrodes
Mixing Chamber
Buffer +
Sample
Stir Bar
D1
Enzyme-Urease being pumped to one of the electrodes (D2)
pH
D1
D2-D1
dpH=a
D2
Tempo (s)
D2
Mixing Chamber
Enzyme- Shown in
Yellow being pumped
into D2
D1
The Reaction Kinetics
mpH
Buffer +
sample
Starter (enzyme /substrate)
D1
Enzymatic
Reaction
D2
Time (s)
What are D1, D2 and mpH?
•
D1 = Difference in mpH measured by 2 electrodes filled with the
same solution (buffer + sample)
•
D2 = Difference in mpH measured by 2 electrodes when Electrode 1
has the same solution of D1 and Electrode 2 has D1 solution +
enzyme
•
mpH = Difference between D2 and D1
mpH is driven by the enzymatic reaction.
Why this technique?
• The technique is based on an enzyme reaction
• The measurement is not affected by interference from ammonium
• Compared to traditional UV methods there is no color, opalescence
and micro-particles interference
• High-tech micro-flow electrodes allow accurate, reproducible
measurements
• These produce a repeatable, reliable, and accurate test method that
has been adopted by many laboratories worldwide
• Eliminate results subjectivity because there is no need to “interpret
results”
• Test results are recorded and can be exported to Excel or printed
out directly from the computer hooked up to the instrument
Comparing with other techniques
Traditional techniques:
Titrators, refractometers, pH-meters
1.
2.
3.
4.
5.
6.
7.
Sample treatment
Colour interference
Preservatives interference
Time consuming
Operator dependency
Limited application panel
Aspecific
Differential-pH
= No sample treatment
= No colour interference
= No preservatives interference
= Faster
= Operator independent
= Wider application range
= Highly specific
Traditional techniques have lower specificity and lower accuracy
Manual Titration
Automated pH meters
and titrators
Refractometers- Cannot
differentiate between
different sugars
Comparing with other techniques
Spectrophotometers (UV)
•
•
•
•
•
•
Differential pH
Sample treatment needed
= No sample treatment
Rather fast
= Equally fast or slightly
slower
Short linearity range
= Wider linearity
Colour interference (auto-treatment) = No interference
Possible Matrix effects
= No effect for tested matrices
Some parameters not reliable
= Reliabile for tested
matrices
An autospectrometer
Comparing with other techniques
IR and FTIR
•
No sample treatment
•
Time/test: 120-400 test/hour
•
Expensive Instrument
instruments
•
Expensive maintenance
Differential-pH
= no sample treatment
= 15 sec/test to 5 minutes
= Medium to lower cost
= Less expensive to maintain
WineScan from FOSS MilkoScan from FOSS
Bentley 2000- For
analysis of fat, protein
and lactose in milk
Comparing with other techniques
HPLC
•
Requires sample treatment
•
Very accurate and precise
•
Time/test 5 to 20 minutes
•
Expensive columns
•
Expensive maintenance
•
Dangerous waste
•
Universal use, versatile
panels
Differential-pH
= No sample treatment
= Equal and comparable
= 15 sec/test to 5 minutes
= Same equipment for all tests types
= Disposable waste
= Limited application/matrices
Quality Indicators and Applications in Milk
Urea
Lactose
L-Lactic Acid
Titratable Acidity
Alkaline Phosphatase
Quality Indicators and Applications in Milk
UREA (ISO Std 14637)
•In most countries urea is included in the milk payment scheme.
•Maximize feed levels
•High urea concentrations  high protein level
•Low urea concentrations  low protein level
•Urea analysis in milk is important because it is related to the animal’s health.
•Levels of urea in milk stay in the range 24-33 mg/100 cc.
LACTOSE
•Lactose occurrs naturally in high concentrations in milk (≈130 mM).
•Also in the payment scheme, in some countries.
•In low-lactose milk products, this is also analysed for consumers with possible
allergy/intolerance.
Quality Indicators and Applications in Milk
L-LACTIC ACID
•Indicator of milk freshness. Fresh milk contains little to no Lactic acid. Milk for
high quality production must contain maximum 30 ppm Lactic acid.
•Process control during fermentation in the yoghurt industries.
•In fermented milk and cheese products, the determination of D and L isomer
forms of lactic acid is very important. Ratio affects taste and aroma.
TITRATABLE ACIDITY
Indicator for milk freshness.
ALKALINE PHOSPHATASE
Indicator for milk freshness.
Alkaline phosphatase enzyme is naturally found in milk. Its content in raw milk
depends on breed, season and lactation phase of the animal.
The enzyme is inactivated at the same temperature conditions where pathogens
micro-organisms are destroyed.
Its absence in milk is an indication of a successful pasteurization.
Quality Indicators and Applications in
Wine, Must and Cider
pH
Sucrose
Glucose
Fructose
Total acidity
Glycerol
Citric Acid
Acetic Acid
L-Lactic acid
L-Malic Acid
Quality Indicators and Applications in
GLUCOSE/FRUCTOSE
Wine
•Control of sugars in grapes and wine or must gives the alcoholic potential
in final product.
TOTAL ACIDITY
•Allows correction if de-acidification is needed.
•Allows monitoring of malic-lactic fermentation.
•Improves the taste of wine.
pH also affects the color of wine.
L-MALIC
•Malic Acid is naturally present in must and develops into L-Lactic Acid in
wine by an enzymatic reaction (Malolactic Fermentation).
•If not controlled, it could also turn into Acetic Acid, which is very
dangerous!).
L-LACTIC
•Monitoring a balance between malic and lactic acid controls taste.
•Affects properties of the final product.
Quality Indicators and Applications in
Wine
ACETIC ACID (the most abundant of volatile acids)
•An unwanted and dangerous compound.
•Corrections are possible at initial stages only!
•Above 1.08 g/l for white wines and 1.20 g/l for red wines, the product is not
allowed to be sold as table wine.
SUCROSE
Sucrose does not naturally exist in wine, thus can be added for special
fermentations (Champagne production) or as an Ethanol developer.
CL-10 Instrument and EC Line Reagents
Summary
• Comprehensive system and reagents for
process and quality control
• Requires no pre-sample treatment
• Ready-to-use reagents
• Fast and reliable
• No sample centrifugation
• Rapid Assay
• Official ISO-IDF method